Abstract

Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations.

Activation of voltage sensor domains (A) VSDs are formed by four transmembrane helices. Upon changes in the membrane potential, the positively charged S4 (blue) moves across the membrane relative to a static S1–S3 bundle (gray), transmitting the electrical signal to a linker peptide (purple). (B) This movement is reported by the measurement of transient currents called gating currents. The time integral of these, the gating charge , can be expressed as the sum of the contributions of the charges of the system. (C) Cartoon depiction of the stepwise activation of the Kv1.2 VSD. From the most resting (ε) to the most activated conformation (α), S4 proceeds in a ratchet-like upward motion in which its positively charged residues jump from a negative binding site to the next. The negative charges of S1–S3 are depicted in red and the ones of the lipid headgroups in yellow. The hydrophobic gasket at the center of the VSD is represented by a green hexagon.

ε/δ-transition in the 2D CV space. (A) The activation step involves a concerted rearrangement of salt bridges between the positive charges of S4, R1–R6 (blue) and the negative charges of S1–S3 (red) and of the lipid headgroups (yellow). (B) Due to the spatial clustering of negative charges, four binding sites can be identified: (i) top phosphate groups (red); (ii) top protein binding site (green); (iii) bottom protein binding site (orange); and (iv) bottom phosphate groups (purple). (C) In this space, during the ε/δ-transition, R1 transfers from site iii to ii and R3 from sites iv to iii.

Free-energy landscape of the ε/δ-transition. (A) Free-energy map in 2D CVR1/CVR3 space. Regions of low free energy are depicted in cold colors (green to blue) and regions of high free energy in hot colors (orange to red). (B) Mapping of the average value of the gating charge as a function of CVR1 and CVR3. (C) Reweighted free-energy profile along . The free-energy profiles at −100 mV and +100 mV were obtained by adding a linear component to the profile obtained under 0 mV using . Starting with the geometrical definition of the two putative activation intermediates ε and δ, we have obtained two larger ensembles of structures corresponding to stable thermodynamic states of the experimental (voltage-clamped) ensemble E and Δ. All free energies are reported in kcal/mol.